Recent advances make NALF a simple and functional platform that will be a serious contender in the POC nucleic acid testing market.
The nucleic acid test (NAT) market has grown significantly in size and diversity in recent years. Generally, nucleic acid tests fall into three categories: those based on direct hybridization and detection using specific nucleic acid probes; those based on signal amplification, where specific probes bind the target sequence and the resulting signal is amplified; and those involving target amplification, typically achieved through enzymatic means. The third category is characterized chiefly by amplification methods that offer enhanced sensitivity, such as the polymerase chain reaction (PCR).
Since its invention in the 1980s, versatile PCR has found a multitude of commercial applications in the life sciences and in vitro diagnostics. More than 20 years later, PCR remains at the center of NAT technology. It is commonplace in the clinical laboratory and essential as a research tool. PCR provides the benchmark for nucleic acid amplification, but many alternative methods have been devised and published that use novel and innovative approaches to achieving amplification and detection of target sequences. Such alternatives include nucleic acid sequence–based amplification, rolling circle amplification, Qß replicase, and simultaneous strand displacement amplification. The majority of these other approaches have application in infectious-disease diagnostics. Although some have proven clinical utility, many may have been developed in an attempt to circumvent patent issues.
More recently, research has concentrated on novel detection techniques and equipment designs that make PCR and other amplification technologies more amenable to current molecular diagnostic requirements. High-throughput automation of sample extraction, amplification, and detection has been the principal focus, primarily driven by blood bank screening for infectious diseases such as HIV and, lately, by genomic screening and single-nucleotide polymorphism (SNP) analysis. Point-of-care (POC) nucleic acid tests have been an elusive goal until recently, largely owing to the complexity of molecular assays and the technical challenges they present with regard to sample preparation and control of assay reproducibility and reliability. Nevertheless, fully integrated POC nucleic acid platforms are emerging. Several multinational companies in the diagnostics and information technology industries have invested significant resources in the development of complex bioengineering strategies centered on microfluidic and bioelectronic sensor technologies. These devices are expensive and currently used only in high-profile applications such as biodefense.
Figure 1. Diagram of a lateral-flow immunoassay rapid test strip.
An alternative, and more cost-effective, approach to POC nucleic acid testing is to use a lateral-flow platform. Lateral-flow immunoassays, which are exemplified by pregnancy test devices, represent a significant portion of today's immunochemical POC market. Their speed, low cost, and simplicity of use make lateral-flow immunoassays the only true point-of-care test for now. These chromatographic devices employ nanoparticles coated with materials that bind to an analyte, such as an antibody or antigen, within a sample. This analyte-nanoparticle complex flows laterally through a series of overlapping membranes until it is captured on an antibody or antigen capture line. A visual result is achieved within a few minutes. Even an unskilled operator can rapidly interpret the test result without any need for complex or expensive equipment (see Figure 1).
Nucleic acid lateral flow (NALF) uses nucleic acid hybridization to capture and detect nucleic acid amplification products in a manner akin to lateral-flow immunoassays. Strips used in the technique do require modified test and capture lines and conjugate pad components. This approach combines the advantages of lateral-flow platforms with those of traditional nucleic acid tests, as described in this article.
Detection with NALF
Figure 2. NALF capture strategies: antibody stripe with labeled amplicon (a), and oligonucleotide probe (b); streptavidin stripe with biotinylated amplicon (c), and biotinylated oligonucleotide probe (d); passive adsorption of BSA-oligonucleotide probe (e), and an unlabeled oligonucleotide probe (f).
Nucleic acids can be captured on lateral-flow test strips in an antibody-dependent or antibody-independent manner (see Figure 2). Antibody-dependent capture, as schematized in the first two drawings in the figure, involves an antibody capture line and a labeled amplicon or oligonucleotide probe of complementary sequence to the amplicon—for example, goat anti-dinitrophenol (DNP) and DNP-label. Antibody-independent alternatives offer more potential for multiplexing, minimize the likelihood of batch-to-batch variation, and may be lower cost. One such method uses noncovalent interaction between two binding partners, exploiting, for example, the high affinity and irreversible linkage between a biotinylated probe or amplicon and a streptavidin line.
A much favored and more simple approach is to immobilize oligonucleotide capture probes directly onto the nitrocellulose lateral-flow membrane. This can be achieved by passive adsorption of a bovine serum albumin–labeled oligonucleotide probe or, preferably, an unlabeled oligonucleotide probe. All of these methods employ standard lateral-flow immunoassay striping equipment and yield strips with long-term stability, often at room temperature.
Figure 3. NALF detection chemistries. In all cases, the oligonucleotide probe may be replaced with an antibody and an appropriately labeled oligonucleotide probe or amplification primer. With an enzyme, an enzyme-probe complex converts substrate into a colorimetric product. With gold, a nanoparticle-probe conjugate provides a visual signal. With Qdot, a Qdot-probe conjugate emits fluorescence. With UPT, a UPT-reporter conjugate is excited at 980 nm, and visible green light is emitted at 500 nm. With a lipsome vesicle, a vesicle-probe conjugate releases dye.
Enzymatic Detection Signals. In theory, any detection chemistry used in lateral-flow immunoassays is applicable also to NALF. The only limitation would be its ability to conjugate the signal molecule of interest to an appropriate antibody or oligonucleotide detection probe. A broad range of NALF detection signals with quantitative and multiplexing capabilities have been reported (see Figure 3).
Reverse-hybridization enzymatic strip assays are the forerunners of today's NALF tests. In these tests, enzyme-labeled probes are hybridized to complementary nucleic acid target species on the surface of a nitrocellulose or nylon membrane. The result is a hapten-antibody-enzyme complex such as, for example, biotin-streptavidin–alkaline phosphatase. Complex wash and substrate-incubation steps are necessary to develop a readable colorimetric signal. Therefore, conversion to conventional lateral flow that will be suitable for nonlaboratory POC NAT applications is a relatively complex matter. Lateral-flow immunoassay–based Fluidics-on-Flex technology (Epocal Inc.; Ottawa, ON, Canada) provides an example. This system incorporates an integral fluidic circuit and pump manifold.
Nanoparticle Detection Signals. Three so-called bead technologies have been used successfully in NALF, namely, colloidal gold, latex, and paramagnetic nanoparticles. Both gold and latex give rise to colorimetric signals visible to the naked eye or semiquantifiable via inexpensive readers. Latex can be manufactured in any color, whereas 2- to 250-nm gold nanoparticles have a characteristic red color that results from surface plasmon resonance.
Gold is the lateral-flow nanoparticle of choice, mainly because of its small size, sensitivity, and robust manufacturing methods.1 It can be conjugated to antibodies and oligonucleotides and labeled with small binding moieties such as biotin or DNP. Gold NALF platforms generally use 30- to 80-nm nanoparticles conjugated to an antibiotin antibody. This gold conjugate is then complexed with a biotinylated amplicon or sequence-specific oligonucleotide detection probe that, when captured by means of an oligonucleotide capture probe or an antibody-hapten-based capture method, yields a signal in the form of line. The optimal size and concentration of the gold nanoparticles used depends on assay specifications, including the application, line intensity, color (cherry red or purple), linear response to target concentration, and uniformity of multiplexed signals.
Paramagnetic NALF is similar to gold NALF but uses 100- to 200-nm superparamagnetic nanoparticles.2 These nanoparticles emit a nonvisual signal when they are subjected to a magnetic field; interpretation requires a specialized reader. Gold and superparamagnetic NALF can detect as little as 1 fmol of synthetic target, sensitivity an order of magnitude greater than that of labor-intensive gel electrophoresis. And superparamagnetic NALF promises further improvement. As with all nanoparticle detection technologies, quality reagents are a key prerequisite. Nanoparticles should be uniform in shape and size and remain free of aggregate.
A number of methods for improving the sensitivity of nanoparticle NALF have been investigated. Detection probes labeled with multiple hapten moieties have been used to form large signal-enhancing lattices by binding specifically to multiple gold nanoparticle conjugates. Combined with real-time PCR, this method can achieve a visual sensitivity similar to that of fluorogenic instrument–based probe methods.3 Also, nanoparticle NALF may be combined with DNA dendrimer signal enhancement, resulting in DNA dendrimers that are branched nucleic acid species with 2 to 900 identical labels per dendrimer. These dendrimers can improve biological assay sensitivities up to 200-fold, depending on the dendrimer size, the application, and the nature of the assay.4
Methods are now available that allow gold and superparamagnetic particles to be coupled to oligonucleotide primers or probes.5 This approach may help to minimize steric hindrance and maximize gold NALF assay sensitivity. Combining such methods with oligonucleotide capture probe immobilization may also eliminate the need for antibody. This antibody-free NALF format can reduce the number of assay components and, in some cases, device cost. With further optimization, this system may improve NALF sensitivity, specificity, and reproducibility. It is also possible to prepare oligonucleotide gold and paramagnetic conjugates that are stable at elevated temperatures. Such conjugates can tolerate thermal PCR cycling conditions, allowing them to be included in amplification reactions.
Emerging Detection Chemistries. More-pioneering detection approaches that draw on liposome and fluorescence methodology are in early stages of development. Liposome nanovesicles constitute a lipid bilayer that can be covalently linked to antibodies and oligonucleotides. These transparent spheres can be used to encapsulate aqueous signals such as dyes in a controlled manner. When employed in NALF testing, oligonucleotide-tagged liposome nanovesicles release dye to yield a visual capture line. A prototype device has been able to detect as few as five viable Cryptosporidium oocysts.6
The use of fluorescence-based lateral-flow immunoassay reporters is on the increase, and several of them have been demonstrated in NALF applications. For example, a dual fluorescein- and biotin-labeled oligo probe has been used to detect single-stranded amplicon generated by cycling probe technology.7 However, the utility of such standard fluorophores is limited by high background fluorescence, the need for a complex reader, and the number of spectrally diverse fluors available for multiplexing.
UPT-NALF is an alternative approach that uses up-converting phosphor reporters, which are approximately 400-nm particles composed of rare earth lanthanide elements that are embedded in a crystal. These particles emit visible light after excitation with infrared radiation in a process called up-conversion. Up-converting occurs only in the phosphor lattice, so autofluorescence of other assay components is virtually nonexistent. UPT has been used to develop rapid prescreening and hybridization-based confirmatory tests for the detection of human papillomavirus type 16, a marker for cervical cancers.8
A third fluorescence approach uses quantum dots, known also as Qdots.9 These nanometer-sized semiconductor nanocrystals have extraordinary optical fluorescence properties that enable them to be as much as a thousand times brighter than conventional dyes. Nanocrystal size determines their color. Their emission profile is narrow and symmetrical, resulting in minimum crosstalk. Qdots are visualized under ultraviolet (UV) light and can be tuned to allow excitation by means of the same long-wavelength UV lamp. The development of water-soluble Qdots that can be conjugated to antibodies (Qdot bioconjugates) has made Qdots amenable to lateral-flow immunoassay applications.10 Initial feasibility testing has employed dot-infused hcG pregnancy tests and is likely to be extended to spectrally multiplexed assays and next-generation NALF applications in the near future.
Detection limits equivalent to or better than those of current gold-standard nucleic acid tests and lateral-flow immunoassays are essential for many POC nucleic acid applications. All of these emerging technologies claim to improve on nanoparticle NALF and enzymatic detection signal sensitivity by two to three orders of magnitude, but they are currently limited by the need for suitable readers or more early-stage development.
Figure 4. Multiplexed NALF, represented in (a) by a schematic showing conversion of an influenza typing panel to that platform, and in (b) by sevenplex detection of synthetic amplicon representing 14 different influenza genotypes: (1) InfA N1 H1; (2) InfA N1 H3; (3) InfA N1 H5; (4) InfA N1 H undefined; (5) InfA N2 H1; (6) InfA N2 H3; (7) InfA N2 H5; (8) InfA N2 H undefined; (9) InfAB negative, H undefined; (10) InfA H1 N undefined; (11) InfA H3 N undefined; (12) InfA H5 N undefined; (13) InfA HN undefined; (14) InfB HN undefined.
Chief areas of imminent POC application of this new NALF technology, and worthy of discussion here, are low-density multiplexed detection and rapid detection of SNP genetic indicators.
Low-Density Multiplexed NALF. Automated high-throughput DNA chip and array technology is well suited for multiplexed detection of very high numbers of samples or probes. However, a need remains for low-density multiplexing platforms for POC detection in the region of 2–25 targets. Low-density multiplexed NALF should fill this niche. This method is designed to detect multiplexed PCR amplicons, as in the Templex assays developed by Genaco Biomedical Products Inc. (Huntsville, AL). A simple multiplexed housed device comprises a single lateral-flow strip, sample port, and conjugate pad, and multiple oligonucleotide capture probe stripes.
A gold-conjugate-based prototype device incorporates seven different target and complementary probes. It has been used to detect synthetic target mixes representing 14 different influenza genotypes in 20 minutes at room temperature (see Figure 4). Preliminary data demonstrate detection of as little as 1 fmol of synthetic nucleic acid sequence, and one-tenth of a standard Templex reaction. A bidirectional housing that incorporates two longer test strips, or a multidirectional housing such as tri- and quad-NALF strips in a tee or cross configuration, can theoretically multiplex much higher numbers of samples. The extent of multiplexing actually is limited by the availability of nonstandard sizes of lateral-flow membranes, the rate and volume of flow through these, and the amount of gold conjugate required. However, preliminary evidence suggests that 24-plexing is feasible with the use of a quad strip format.
Figure 5. SNP NALF detection strategies. (a) In competitive allele-specific short oligonucleotide hybridization, biotinylated and unlabeled mutant and wild-type sequence-specific hybridization probes compete at the SNP site, resulting in sequence-dependent presence or absence of signal at a streptavidin capture line. (b) Alternatively, chimeric PCR primers that incorporate hexapet tags are used to synthesize PCR amplicons in an allele-specific manner, with the resulting amplicons being captured at room temperature on complementary striped hexapet oligonucleotide probes.
The major hurdle to be overcome in multiplexed NALF is the nonspecific signal inherent in NAT platforms comprising large numbers of probes and amplification primers. Minimization of this can be achieved through careful optimization of primer and probe sequences, concentrations, and positioning of capture lines.
SNP NALF. Single-nucleotide polymorphisms are important indicators of human genetic disease, strain genotypes, and drug resistance. A number of rapid SNP NALF detection techniques amenable to POC are in development.
Most PCR-based SNP diagnostics use a single primer pair to make amplicons with variable internal regions containing one or more single-base mismatches. One such approach involving NALF technology is competitive allele-specific short oligonucleotide hybridization (CASSOH).11 This method discriminates at the capture level by means of biotinylated sequence-specific hybridization probes designed to contain the SNP base of interest. Competition between labeled and unlabeled mutant and wild-type probes results in the presence or absence of signal at a streptavidin capture line in a target-sequence-dependent manner (see Figure 5).
A disadvantage of the CASSOH system is the requirement for separate reactions for each target sequence and a postamplification temperature ramping. However, an alternative approach has been devised that uses allele-specific PCR and chimeric PCR primers that incorporate hexameric repeat tags termed hexapet tags.12 These tags, designed to exhibit minimal cross-reactivity, have been used to demonstrate specific hybridization-based capture of amplicon at room temperature using NALF strips striped with complementary hexapet tag sequences (see Figure 5b). This system discriminates between alleles at the amplification level, which brings its own disadvantage: multiple allele-specific primers are required.
Figure 6. Duplex detection of SNPs using hybrid nucleic acid probes in a platform developed by BBInternational (Cardiff, UK). (a) In the schematic representation, SNP-specific oligonucleotide detection probes (stripes) capture single-stranded PCR amplicon. The antibiotin-gold conjugate (red circle) and biotinylated detection probe (green circle) are immobilized in a dry-conjugate-pad format. (b) The test images reveal housed full-dipstick discrimination of factor II wild-type and mutant genotype sequences (C = wild type, G = mutant).
BBInternational (Cardiff, UK) is developing a noncompetitive PCR NALF platform that discriminates at the detection level. Short immobilized hybrid nucleic acid probes are designed to have carefully optimized melting temperatures. This system has demonstrated discrimination at room temperature without the need for allele-specific primers, and detects asymmetric PCR amplicon or amplicon that has been rendered single-stranded by lambda exonuclease enzyme digestion (see Figure 6).
All of these SNP-based NALF platforms potentially can be used to multiplex detection of two or more SNPs in a single device. In all cases, the key to success is careful primer and probe design.
Methods are available that allow for the manufacture of generic NALF strips.13 Such strips include a bridge oligo with a first region complementary to a generic striped oligonucleotide probe, along with a second sequence-specific region complementary to the amplicon of interest (see Figure 7). Detection of different target sequences requires redesign of the bridge oligo, but the same generic NALF strip can be employed, which theoretically minimizes strip redesign.
An extension of this generic approach is the development of combination lateral-flow immunoassay strips that utilize nucleic acid base pairing to facilitate detection of immunochemical targets (Figure 7b). Detection involves a generic striped oligonucleotide probe capturing a complementary oligonucleotide–human antibody conjugate. The analyte of interest is sandwiched between the capture conjugate and a cross-reacting antigen-label conjugate.
NALF Assay Design
Figure 7. Generic NALF strips take two basic forms. (a) In a bridging-probe NALF, detection is facilitated by a bridging oligonucleotide probe with a first portion complementary to a labeled PCR product and a second portion complementary to a striped capture oligonucleotide. (b) The immunochemical-NALF combinatorial sandwich assay approach involves bringing the patient antibody (red circle) between an antigen-labeled conjugate detection reagent and an antihuman antibody–oligonucleotide conjugate.
Most thermal and isothermal NAT amplification technologies can be readily converted to NALF by adaptation of probes and labeling moieties. In many cases, amplification and detection probe sequence redesign is unnecessary; thus, development time and cost are minimized, and so may be the time required to secure regulatory approval. The incorporation of amplification and detection controls is also relatively straightforward, just a matter of including one or more additional capture lines and complementary probes and/or primers.
Designers of NALF assays must take into account general lateral-flow immunoassay design criteria that influence assay sensitivity and nonspecific signal.14 Nucleic acid–compatible sample and conjugate pads and blocking materials must be chosen. For example, nucleic acids will attach to glass-fiber materials, causing sample retention and inhibiting probe release. However, these effects can be minimized through the application of novel pad materials and blocking strategies.
Nitrocellulose membrane pore size and flow rate also are key, as there is a trade-off between assay time and efficient target-probe hybridization. NALF detection is not particularly prone to sample effects, because the sample often is diluted in upstream processes such as amplification. Gold NALF assays can detect amplification reactions involving blood products and crude bacterial cell lysates without significant signal inhibition. Thus, unlike with lateral-flow immunoassays, complex sample-pad materials for upstream processes such as blood separation are not always necessary.
Manufacturing Implications and Limitations
Future NALF devices are likely to include dry-format amplification and detection reagent technology and versatile lateral-flow housing designs. Antibody- and oligonucleotide-nanoparticle conjugates can already be supplied in dry, soaked conjugate pad, and sprayed reagent line formats. Technology also is available to combine PCR and nanoparticle NALF components into a dry pellet that can be reconstituted upon sample addition.
Standard chromatographic lateral-flow materials are used for NALF strip manufacture, and strong similarity between NALF probe and antibody immobilization methodologies allows standard automated lateral-flow manufacturing equipment to be employed. Equipment costs, therefore, are minimized. Also, there is no need to retrain production staff. Unmodified oligonucleotides are significantly cheaper than striped antibodies, and oligonucleotide-striped membranes have long-term stability at room temperature. Successful NALF detection chemistries will be those operated and stored at room temperature.
Figure 8. A diverse range of lateral-flow housing designs.
Today's lateral-flow immunoassay designs can be extremely elaborate, incorporating novel functions such as separate sample and buffer ports; buffer bags and blister packs that entail piercing or wick insertion; multidirectional flow; multiple analyte detection; and moving parts (see Figure 8). Standard injection-molded housings often hide complex mechanisms such as wash steps behind simple push-button fascias. Similar approaches are being used to develop novel closed-tube NALF housing designs that will meet future POC needs in the clinical, environmental, and food diagnostic sectors. BBInternational, for example, is developing a housing that allows cross-contamination-free transfer of a PCR reaction to a NALF test strip.
NALF assay detection is rapid. Detection has been achieved in as little as 5 minutes, the speed depending on the application and the detection scheme. A major future challenge for manufacturers will be to incorporate rapid, simple, and relatively inexpensive upstream sample extraction and amplification processes that will reduce the overall time to result. Future NALF devices are likely to integrate emerging technologies that shrink these process bottlenecks to a matter of minutes. The RapidCycler 2 instrument from Idaho Technology Inc. (Salt Lake City) already achieves amplification in 15 minutes, but it comes with a high price tag. Microfluidic PCR devices of the future may prove to be simpler, less expensive, and more amenable to NALF. Prototypes are emerging that have no moving parts or complex in situ components such as pumps, valves, or wells, and that perform PCR in less than 5 minutes with the same yield as a typical 90-minute thermal cycler protocol.
Similarly, rapid and expensive benchtop extraction equipment is reaching the market. The PlasmaGen APR-510-S from Atmospheric Glow Technologies Inc. (Knoxville, TN), for example, provides one-step extraction of DNA or RNA from a variety of dry sample matrices in 1 to 2 minutes, and the QuickGene-810 of Fuji Photo Film Company, Ltd. (Tokyo), isolates DNA and RNA from whole blood in 6 minutes. Another approach uses rapid cycles of hydrostatic pressure to extract biological material. All of these methods yield high-quality nucleic acid from low-abundance samples and are potentially NALF-compatible. However, a need for simple, low-cost alternatives remains.
A variety of manufacturers offer magnetic-bead systems that do not require centrifugation, but these systems generally do require open-tube transfer from the lysis tube to the amplification vessel. Some progress has been made with the development of chaotropic archiving materials. These so-called papers contain chemicals that lyse cells, denature proteins, and protect nucleic acids from nucleases, oxidation, and UV damage. Unfortunately, unfavorable wash steps are necessary. Tubes coated with a solid-phase matrix that irreversibly binds nucleic acid, allowing extraction and PCR in the same reaction vessel, are another step in the right direction.15 Future solutions may lie in improved amplification technologies that can tolerate cruder sample extracts.
Some elements of the NALF technologies described above draw on skills known in the art, while others are patent-protected or based upon carefully guarded know-how. When choosing a NALF developer, it is important to consider the impact that early immunochromatographic lateral-flow patents may have on the business arrangement. BBInternational has negotiated an agreement with Inverness Medical Innovations that offers protection for contract development and manufacturing customers on a selection of lateral-flow patents within the Inverness portfolio.
Recent technological advances in nanoparticle and alternative detection chemistries, along with the development of flexible platforms that allow multiplexing and SNP detection, make NALF a serious contender for a place in the future nucleic acid POC diagnostic test market. This rapid, simple, and inexpensive detection methodology has potential for application in rapid infectious-disease strain typing, human genetic disease diagnosis, and environmental field-site testing. Development of sensitive and accurate NALF-type POC tests will most likely be achieved through interdisciplinary partnering between molecular biology specialists, experienced immunochemical lateral-flow assay developers and manufacturers, and experts in sample extraction, amplification processes, and equipment design. This approach will allow full exploitation of this flexible technology and bring the industry a step closer to true POC nucleic acid tests.
|(Left to right) Joanna Seal, PhD, is senior project leader of the nucleic acid division, Helen Braven, PhD, is a project leader in research and development, and Paul Wallace, PhD, is technical director at British Biocell International (Cardiff, UK). The authors can be reached at email@example.com, firstname.lastname@example.org, and email@example.com, respectively.
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